Formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing Klebsiella pneumoniae in vitro in Kenya

Background The development of alternative control measures, such as phage therapy or adjunctive therapy, is urgently needed to manage the dissemination of carbapenemase-producing Klebsiella pneumoniae. Objective This study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of carbapenemase-producing K. pneumoniae clinical isolate in vitro in Kenya. Methods The study was conducted from February 2021 to October 2021 at the Institute of Primate Research, Nairobi, Kenya. Phage cocktails were formulated based on the morphology and biological properties of precipitated Klebsiella phages. The efficacy of individual bacteriophages and phage cocktails as well as their combination with antibiotics were determined for their inhibitory activity on carbapenemase-producing K. pneumoniae (KP20). Results The precipitated bacteriophages were members of Myoviridae, Siphoviridae and Podoviridae. Regarding the evaluation of the phage cocktails, the absorbances at 600 nm of the bacterial culture treated with the two-phage cocktail (2φ MA) ranged from 0.173 to 0.246 at 16 h and 20 h whereas it peaked from 2.116 to 2.190 for the positive control. Moreover, the results of the adjunctive therapy showed that the optical density at 600 nm of the bacterial culture treated with 2φ MA was 0.186 at 24 h post-incubation time while it was 0.099 with the bacterial culture treated with imipenem in combination with 2φ MA. Conclusion This study demonstrated that the two-phage cocktail in combination with imipenem was able to synergistically delay the increase in carbapenemase-producing K. pneumoniae growth in vitro.


Introduction
The development of alternative control measures is urgently needed to manage the dissemination of carbapenemase-producing (CP) Klebsiella pneumoniae because of the decrease in the effectiveness of antibiotic drugs. 1,2 Bacteriophage therapy has, therefore, been proposed as an alternative strategy in controlling multidrug-resistant (MDR) bacterial infections, including MDR K. pneumoniae clinical isolates. 3,4 Some of the beneficial effects of bacteriophages over antibiotics include their abundance, host specificity and exponential replication. 5 However, the narrowness of phage host range and the ever emergence of novel pathogen variants will at minimum represent some limitations for phage therapy. 6,7 This challenge can be managed by formulating phage cocktails that contain different phages infecting one species or by combining phages with antibiotics, which may result in a broad spectrum of activity. 8 The characterisation of therapeutic phages in terms of their biology and bactericidal

Formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing Klebsiella pneumoniae in vitro in Kenya
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http://www.ajlmonline.org Open Access activity is mandatory before any preclinical trials because the application of non-characterised lytic bacteriophages may cause undesirable virulence as an adaptive response to bacteriophage infection. 5,9 In other words, the development of effective strategies, such as phage growth parameters, host range spectrum, the presence of lytic proteins, formulation of phage cocktail and phage synergistic interaction with antibiotic to enhance phage efficacy, is a key prerequisite for optimal treatment of MDR infections caused by K. pneumoniae. Several studies have reported the advantages and drawbacks of phage cocktails in treating MDR bacteria. 10,11 Unfortunately, bacteriophages employed as cocktail or adjunctive therapy have not been well investigated in the African continent to effectively control MDR bacterial infections. 12 This study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of CP K. pneumonia clinical isolate in vitro in Kenya.

Ethical considerations
This study was not human or animal research. However, clearance was issued by the Nairobi City Water & Sewerage Company Limited (NCWSC/HR/TRG.14/Vol.8/14/MMM/ ak) for collection of waste water samples.

Bacterial isolate collection, phage isolation and purification
The study was conducted from February 2021 to October 2021 in the Phage Biology Research Laboratory at the Institute of Primate Research, Nairobi, Kenya. The clinical isolate of CP K. pneumoniae (KP20) was obtained from the stock of the recently published data in Kenya. 13 Bacteriophages were isolated from waste water samples of Dandora Estate Sewage Treatment Works (Ruai) and Rongai effluent, Nairobi, Kenya. They were purified from waste water samples through spot assay and double-layer plaque method as described, previously. 14,15

Determination of minimum inhibitory concentrations for KP20
The minimum inhibitory concentrations of a panel of antibiotics including carbapenems for KP20 were determined using VITEK 2 Systems version 9.2 and AST-XN05 (bar code 1481424403205844) card (BioMerieux, Inc., Hazelwood, Missouri, United States), according to the manufacturer's instructions. The detailed procedure is described in online Supplementary Method 1.

Bacteriophage propagation and precipitation
Phage lysate (≥ 10 8 PFU/mL or 10 9 PFU/mL) was propagated in large volume, concentrated and cleaned up in the presence of sodium chloride and polyethylene glycol as described elsewhere. 14,16

Morphological characterisation of precipitated bacteriophages
The morphological characterisation of the precipitated Klebsiella phages was conducted at the University of Leicester, United Kingdom. Negative staining with 1% (weight/ volume) uranyl acetate on 3 mm carbon-coated copper grids of the precipitated Klebsiella phages was conducted at the University of Leicester and the bacteriophages were visualised with a transmission electron microscope (JEOL UK Ltd., Welwyn Garden City, United Kingdom). 17 Bacteriophages were then classified according to their morphology. 18

Bacteriophage host range determination
A spot assay was conducted to determine the host range of precipitated bacteriophages and phage cocktails as described by Kutter. 19 Bacterial strains tested in our study included reference strains and MDR bacterial strains reported by Michodigni et al. 13 and described in online Supplementary Methods 2.

One-step growth experiment
A one-step growth experiment for determination of latent period, and burst size of precipitated bacteriophages, was performed as described elsewhere, with some modifications. 20 Exponential-growth-phase culture of CP K. pneumoniae (≈ 2.98 × 10 8 CFU/mL) and phage precipitate (≈ 10 9 -10 10 PFU/ mL) were mixed with bacterial suspension to obtain a multiplicity of infection (MOI) of 0.1. 14 The suspension was incubated at 37 °C in a shaking incubator for 10 min at 120 rotations per minute following with centrifugation. The pellet was then suspended in 7 mL of fresh tryptic soy broth and incubated at 37 °C in a shaking incubator at 125 rotations per minute. At 3-min intervals, from 0 min to 36 min, 500 μL of the suspension was taken and phage titres were estimated. 20,21 Formulation of phage cocktails and evaluation of their in vitro activity on carbapenemaseproducing Klebsiella pneumoniae

Evaluation of inhibitory activity of phage cocktails in combination with antibiotics on carbapenemase-producing Klebsiella pneumoniae in vitro
The inhibitory activity of the phage cocktail alone, and in combination with imipenem or tigecycline was assessed on KP20 at its optimal exponential growth (OD 600 ≈ 0.

Data analysis
A two-tailed t-test was performed to determine the significance levels of phage cocktails in combination with select antibiotics using GraphPad Prism version 9.2.0. (GraphPad Software, San Diego, California, United States). A p < 0.05 was considered as significantly different.

Minimum inhibitory concentrations of antibiotics and detected carbapenemase genes
The minimum inhibitory concentrations of imipenem and tigecycline to KP20 were 4 μg/mL and 1 μg/mL (Table 1). We previously reported the presence of extended-spectrum beta-lactamase (ESBL) genes (blaTEM and blaOXA) and carbapenemase genes (blaNDM-1, blaIMP). 13 The ESBL and carbapenemase genes produced by KP20 are indicated in Table 1.

Morphological characteristics of precipitated Klebsiella phages
A transmission electron microscope showed that the bacteriophages included in this cocktail were all tailed phages ( Figure 1). Among the three imaged bacteriophages, two were myoviruses (CPRSA and CPRSB) and one was a podovirus (ESBLA).

Precipitated Klebsiella phages growth characteristics
The burst sizes of the precipitated phages in the cocktail were 610 (

Host range of Klebsiella phage lysates
Klebsiella phages CPRSA, CPRSB and ESBLA were used for the formulation of the two-phage cocktail (2φ MA) and the three-phage cocktail (3φ MB). The two-phage cocktail comprised CPRSA and CPRSB while the three-phage cocktail consisted of CPRSA, CPRSB and ESBLA. The two-phage cocktail was the combination of two bacteriophages belonging to the family Myoviridae while the three-phage cocktail was the combination of 2φ MA and one belonging to the family Podoviridae. The results of the host range analysis showed that both the individual phages and phage cocktails (2φ MA and 3φ MB) were active on bacterial strains belonging to Klebsiella species. The three carbapenem-resistant K. pneumoniae were susceptible to only one individual phage, the Klebsiella phage CPRSA. The two-phage preparations were also active on the three CP K. pneumoniae strains, in addition to K2, which was described as an extended-spectrum beta-lactamase producer in our previous study.  (Table 3). Significant difference (p = 0.01) was observed between the two treatments at MOI 0.001.

Interactive properties between the phage cocktails and antibiotics combinations in inhibiting the growth of carbapenemaseproducing Klebsiella pneumoniae in vitro
The in vitro studies showed that the same phage cocktails (2φ MA and 3φ MB) maintained their bactericidal activity after 24 h of incubation at the lowest phage concentration (MOI 0.001) (Figure 3a). At different timepoints of 18 h and 24 h, the optical density at 600 nm of the bacterial culture        Table 4. The results of bacterial cultures treated with phage cocktail alone, and in combination with imipenem, were statistically significant compared with the result of bacterial culture treated with only imipenem (p = 0.02). No statistical difference was observed between the results of the bacterial culture treated with a combination of phage and antibiotic and those of bacterial culture treated with tigecycline alone. A significant difference (p = 0.04) was observed between the bacterial culture treated with 2φ MA at MOI 0.001 and the adjunctive therapy (bacterial culture treated with IMP2 + 2φ MA3).

Discussion
This study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of the KP20 clinical isolate. On one hand, the phenotypic results revealed that the precipitated phages obtained in this study had lytic activity with high titers and they were related to tailed phages. The latter are generally divided into three families including Myoviridae with contractile tails consisting of a sheath and a central tube (25% of tailed phages), Siphoviridae with long and noncontractile tails (61%), and Podoviridae having short tails (14%). 23 Klebsiella tailed phages identified in our study belonged to the family of Myoviridae and Podoviridae. 24 Previous studies reported similar observations regarding the morphological characters of Klebsiella phages. 25 On the other hand, this study showed that one of the precipitated phages (CPRSA) had the shortest latency period (9 min) and high burst size (610). Our findings were supported by Horváth et al. in 2020. The authors reported that their isolated Klebsiella phage had a relatively short latency period of 18 min and its burst size was ~220 phage particles per infected bacteria. 20 Indeed, a high burst size is key to achieve a productive adsorption and replication of bacteriophages and to reach the benefits that phages could have in comparison with antibiotics while a short latency period is recommended from a phage therapy perspective. 26 Both the individual bacteriophages and phage cocktails were not susceptible to the reference bacterial strains tested in this study and, hence, displayed high specificity for their host bacteria, which were KP20 clinical isolates. Previous studies on host ranges of Klebsiella phages reported similar results. 27,28,29 The major consequence of phage host specificity is that it demands an appropriate diagnosis of the bacteria involved in the infection before initiation of phage treatment. 7,30 Nevertheless, this narrowness of phage host range to the strains of the same bacterial species could limit its lytic activity on microbiota. 7,30 The phage host range is indeed affected by a number of factors including the absence of required accessory proteins for phage adsorption, restriction-modification and clustered regularly interspaced short palindromic repeats (CRISPR) systems. 31,32 Despite their narrow host range, bacteriophages with lytic activity may still be useful in phage therapy and the use of bacteriophages as cocktails for adjunctive treatment can represent a highly attractive strategy. 33 Besides the choice of bacteriophages with different bacterial cell wall receptor recognition sites in formulating a phage cocktail, the most important criterion for successful phage cocktail preparation also includes the compatibility of bacteriophages in mixtures. 22 Therefore, our study demonstrated that the mixture of two phages (2φ MA) was able to significantly delay the resurgence of bacterial cells in culture, as compared to the application of individual phage or the mixture of three phages (3φ MB) (p = 0.02). Our result was in line with the previous data published on phage cocktail efficacy. 11 This synergistic activity of two-phage cocktail over the mixture of three phages in our study suggested that the individual phages might have employed different receptors to adsorb to the bacterial cells and, hence, effectively inhibited the bacterial growth. Some authors have reported that one phage in their two-phage cocktail used an outer membrane protein (OmpC) as a receptor, and another one employed a lipopolysaccharide component as its receptor to effectively control bacterial resistance. 34,35 Moreover, the mixture of many phages may be less effective in the absence of identification of specific bacteriophage receptors because the same phages might share the same receptors and interfere with one another. 36,37 The adjunctive treatment (IMP2 + 2φ MA3) significantly inhibited the growth of KP20 in vitro compared to the twophage treatment alone (p = 0.04). At MOI 0.001, the twophage cocktail combined with imipenem had significantly lysed the bacterial cell compared to the two-phage cocktail. This statistical difference might be related to the beneficial effect of bacteriophage treatment in adjunctive therapy. Our finding contradicted the study of Pacios et al., who indicated the inability of phage-imipenem combination to kill imipenem-resistant isolate harbouring OXA-245 b-lactamase. 38 This divergence might be related to the use of a single lytic phage in adjunctive treatment instead of phage cocktail. Furthermore, an antibacterial effect was also observed between the phage cocktails in combination with the most effective antibiotic (tigecycline) without significant difference (p = 0.99). A number of studies have reported the positive effect of bacteriophages in combination with antibiotics. 39 This synergistic activity between phage cocktail in combination with imipenem might have been due to the sensitivity of the bacterial strains to the select antibiotic after bacteriophage action. Indeed, it was reported in a previous study that phage-resistant bacterial strains are more susceptible to antibiotics and the rate of their growth is slower in comparison with wild bacterial strains. 40 Our study also demonstrated the efficacy of phage cocktail in the absence of phage receptor analysis in formulating phage cocktails. Interestingly, this study pointed out the repurposing of imipenem using phage cocktail therapy, and further encourages the repurposing of the efficacy of Food and Drug Administration-approved antibiotics for acceptance of phage therapy worldwide, especially in Africa.

Limitations
The limitation of this study was the absence of genomics, which could enable us to further conclude on the taxonomic classification of the bacteriophages. The conclusions were made based on a single carbapenem-resistant strain, which was also a limiting factor in our study due to the high host specificity of phages, and the diverse K. pneumoniae strain types and resistance mechanisms which may affect the utility of the adjunctive therapy. The potential impact of phages on the normal microbiota was not considered in the current study, especially given that the phage cocktails might have activity against K. pneumoniae strains other than those that are carbapenem-resistant, and E. coli strains.

Conclusion
This current study revealed the presence of lytic tailed Klebsiella phages belonging to the family Myoviridae, Siphoviridae and Podoviridae in Nairobi sewage systems with relatively short latent periods and optimal burst sizes, indicating their therapeutic potential in composing phage cocktails and synergistic interaction in combination with non-sensitive antibiotic (imipenem) against carbapenem-resistant K. pneumoniae clinical isolate in vitro.